【作者】 李姣；

【导师】 杨青； 刘磊；

【作者基本信息】 湖南农业大学 ， 临床兽医学， 2017， 硕士

【摘要】 目的:探索支链脂肪酸(branched chain fatty acids,BCFA)iso-15:0和iso-18:0的降脂作用及其机制,为支链脂肪酸的生物活性研究和开发提供理论依据,以期为非酒精性脂肪肝病(NAFLD)的天然药物或保健食品研发提供新思路。方法:以人正常肝细胞系(L02)为实验材料,用FFA(free fatty acid,油酸:棕榈酸=2:1)体外模拟建立人单纯性肝甘油三酯沉积模型,采用不同浓度的iso-15:0或iso-18:0对L02细胞进行处理,通过噻唑蓝(MTT)法检测L02细胞增殖率,GPO-PAP法测定细胞内甘油三酯(TG)含量,油红O染色法测定细胞内脂质水平以监测两种BCFA的作用效果;并进一步采用荧光定量PCR法和Western Blotting法分别从m RNA和蛋白水平检测脂类代谢关键因子(包括PPAR-α、CPT1、CPT2、ACOX1、SREBP-1c、ACC、m TOR、FASN m RNA和PPAR-α、CPT1、SREBP-1c、ACC、AMPK、P-AMPK蛋白)在上述处理细胞模型中的变化,以探讨两种BCFA的作用机制。实验所得数据均采用SPSS19.0进行分析,以P<0.05为显著差异。结果:(1)用1 m M FFA处理L02细胞24 h时能极显著提高细胞内TG的含量(P<0.01),且对细胞增殖无明显影响,符合单纯脂肪变性模型的特征。(2)两种支链脂肪酸iso-15:0和iso-18:0均能降低单纯脂肪变性模型L02细胞内脂质的含量。(3)iso-15:0和iso-18:0对脂质的下调主要通过下调肝脏脂肪合成相关酶SREBP-1c、ACC、FASN的m RNA表达水平,以及SREBP-1c和ACC的蛋白表达水平,并上调脂肪分解关键酶PPAR-α、CPT1、CPT2、ACOX1的m RNA表达水平,以及PPAR-α、CPT1和AMPK的蛋白表达水平而实现。结论:支链脂肪酸iso-15:0和iso-18:0在体外有显著的降脂作用,其作用机制可能是通过降低肝脏细胞部分脂肪合成关键酶包括SREBP-1c、ACC、FASN的表达,同时提高部分脂肪分解关键酶包括PPAR-α、CPT1、CPT2、ACOX1的表达水平,并激活AMPK能量代谢通路,使细胞内脂肪合成速度减慢,从而加快脂肪分解代谢而实现。更多还原

【Abstract】 Objective: It was aimed to explore the lipid-lowering effect of two branched chain fatty acids(BCFA),iso-15:0 and iso-18:0,and study the mechanisms involved so as to deepen our understanding of the biological activity of BCFA and their application,and also provide new clues for the development of natural drugs or health food for nonalcoholic fatty liver disease(NAFLD)control.Methods: To establish a cell model for investigating hepatic steatosis in vitro,L02 cells were exposed to a mixture of free fatty acids(FFA)(oleates: palmitates=2:1).The morphology of lipid droplets,triglyceride(TG)level and cell proliferation rate were evaluated by using Oil Red O staining,GPO-PAP and MTT methods,respectively.Then established cell model were treated with different concentrations of iso-15:0 and iso-18:0BCFA,respectively,and the TG-lowing effects were observed by determination of total lipid content and TG level.Furthermore,m RNA levels of several key factors of lipid metabolism(PPAR-α,CPT1,CPT2,ACOX1,SREBP-1c,ACC,m TOR,FASN m RNA)and protein levels of PPAR-α,CPT1,SREBP-1c,ACC,AMPK and P-AMPK were examined by using q PCR and Western Blotting methods to explore the molecular mechanisms of iso-15:0 and iso-18:0.The data were statistically analyzed using software SPSS 19.0,and P < 0.05 was considered to be statistically signi?cant.Results:(1)After treated with 1 m M FFA for 24 h,L02 cells showed a markedly increased level of TG without significant changes of cell proliferation rate,which indicated that the hepatic steatosis cell model was successfully established.(2)Both iso-15:0 and iso-18:0BCFA could decrease the lipid content in the established cell model.(3)iso-15:0 and iso-18:0 BCFA played their roles through suppression of SREBP-1c,ACC and FASN.In addition,incubation with iso-15:0 and iso-18:0 BCFA up-regulated the expression of lipolytic enzyme,CPT1,CPT2,PPAR-α,ACOX1,AMPK.Conclusions: Iso-15:0 and iso-18:0 can significantly decrease the level of TG in a hepatic steatosis cell model.Suppression of hepatic cell fatty synthase,SREBP-1c,ACC and FASN etc,and up-regulation of the expression of catabolismenzyme,like CPT1,CPT2,PPAR-α,ACOX1 etc,may account for the molecular mechanism for the biological activity.Also,iso-15:0 and iso-18:0 are able to activate the AMPK pathway,leading to a decrease in the rate of lipid synthesis but an increase in the rate of breakdown of lipids in cells.更多还原